Genetic polymorphism in the Zf9 gene linked to inappropriate scarring or fibrosis

ABSTRACT

The present invention relates to an in vitro method for diagnosing or detecting a predisposition to a condition at least partially characterised by inappropriate fibrosis or scarring (e.g. Dupuytren&#39;s Disease). The method comprises examining the ZF9 gene, and regulatory elements thereof, derived from a subject of interest to detect the presence of a genetic poylmorphism or mutation in said gene which is linked with the condition.

This application is the US national phase of international applicationPCT/GB02/00847 filed 01 Mar. 2002, which designated the US.

The present invention relates to methods for the genetic testing ofsamples to determine the presence of polymorphisms in the Zf9 gene thatare linked to a genetic predisposition to develop conditions at leastpartially characterised by inappropriate scarring or fibrosis.

A scar is an abnormal morphological structure resulting from a previousinjury or wound (e.g. an incision, excision or trauma). Scars arecomposed of a connective tissue which is predominately a matrix ofcollagen types 1 and 3 and fibronectin. The scar may consist of collagenfibres in an abnormal organisation (as seen in normal scars of the skin)or it may be, an abnormal accumulation of connective tissue (as seen inscars of the central nervous system or pathological scarring of theskin).

Scarring is a usual consequence of the healing process in most adultanimal and human tissues. In the skin scars may be depressed below thesurface or elevated above the surface of the skin. Hypertrophic scarsare a more severe form of scarring that can arise in certain conditionsor certain individuals. Hypertrophic scars are elevated above the normalsurface of the skin and contain excessive collagen arranged in anabnormal pattern. A keloid is a form of pathological scarring which isnot only elevated above the surface of the skin but also extends beyondthe boundaries of the original injury. In a keloid there is excessiveconnective tissue which is organised in an abnormal fashionpredominantly in whorls of collagenous tissue.

There are believed to be genetic predispositions to forminginappropriate, and particularly pathological, scarring (e.g.hypertrophic scars and keloids). For instance, Africo-Carribean andMongoloid races are particularly prone to developing keloid scars.

There are numerous medical situations where scar formation represents aproblem. Examples of such situations are scars of the skin whereexcessive scarring may be detrimental to tissue function andparticularly when scar contracture occurs (for instance skin burns andwounds that impair flexibility of a joint). The reduction of scarring tothe skin when cosmetic considerations are important is also highlydesirable. In the skin, hypertrophic or keloid scars particularly inAfrico-Caribbean and Mongoloid races) can cause functional and cosmeticimpairment and there is a need to prevent their occurrence. Scarringresulting from skin grafts in both donor sites and from the applicationof artificial skin can also be problematic and need to be minimised orprevented. Given the importance of scarring in such situations, it willbe appreciated that there is a need to be able to test a subject toinvestigate whether or not they will be susceptible to developinginappropriate scarring.

As well as scars of the skin, internal scarring or fibrosis can behighly detrimental and specific examples include:

(i) Within the central nervous system, glial scarring can preventneuronal reconnection (e.g. following neuro-surgery or penetratinginjuries of the brain).

(ii) Scarring in the eye can be detrimental. In the cornea, scarring canresult in abnormal opacity and lead to problems with vision or evenblindness. In the retina, scarring can cause buckling or retinaldetachment and consequently blindness. Scarring following wound healingin operations to relieve pressure in glaucoma (e.g. glaucoma filtrationsurgery) results in the failure of the surgery whereby the aqueoushumour fails to drain and hence the glaucoma returns.

(iii) Scarring in the heart (e.g. following surgery or myocardialinfarction) can give rise to abnormal cardiac function.

(iv) Operations involving the abdomen or pelvis, often result inadhesion between viscera. For instance, adhesions between elements ofthe gut and the body wall may form and cause twisting in the bowel loopleading to ischaemia, gangrene and the necessity for emergency treatment(untreated they may even be fatal). Likewise, trauma or incisions to theguts can lead to scarring and scar contracture to strictures which causeocclusion of the lumen of the guts which again can be life threatening.

(v) Scarring in the pelvis in the region of the fallopian tubes can leadto infertility.

(vi) Scarring following injury to muscles can result in abnormalcontraction and hence poor muscular function.

(vii) Scarring or fibrosis following injury to tendons and ligaments canresult in serious loss of function.

Related to the above is the fact that there are a number of medicalconditions known as fibrotic disorders in which the development ofexcessive fibrotic tissue leads to pathological derangement andmalfunctioning of tissue. Fibrotic disorders are characterised by theaccumulation of fibrous tissue (predominately collagens) in an abnormalfashion within the tissue. Accumulation of such fibrous tissues mayresult from a variety of disease processes. These diseases do notnecessarily have to be caused by surgery, traumatic injury or wounding.Fibrotic disorders are usually chronic. Examples of fibrotic disordersinclude cirrhosis of the liver, liver fibrosis, glomerulonephritis,pulmonary fibrosis, cystic fibrosis, scleroderma, myocardial fibrosis,fibrosis following myocardial infarction, central nervous systemfibrosis following a stroke or neuro-degenerative disorders (e.g.Alzheimer's Disease), proliferative vitreoretinopathy (PVR) andarthritis.

As is the case for scarring, it is believed that their is a geneticinfluence on the development of many fibrotic disorders or the severitythereof. In some instances, the genetic influence may be directlyresponsible for the development of the disorder whereas for otherfibrotic disorders there may be a genetic factor that influencesfibrotic development which is secondary to the primary cause of thedisorder (e.g. in cystic fibrosis).

One example of a disorder that involves fibrosis, and is also believedto be influenced by genetics, is Dupuytren's disease (DD).

DD is a nodular palmar fibromatosis causing progressive and permanentcontracture of the digits. DD is often familial and is common inindividuals of Northern European extraction. In excess of 25% of malesof Celtic races over 60 years of age have evidence of DD and it isconsidered to be one of the most common heritable disorders ofconnective tissue in Caucasians.

Autosomal dominance with variable penetrance has been proposed as thelikely mode of inheritance for DD. However no single gene has so farbeen identified in the literature as being responsible for thecondition. Furthermore it is unclear whether DD is a complex oligogeniccondition or a simple monogenic mendelian disorder. Accordingly theidentification of susceptible genetic loci would provide an idealapproach to unravelling the hereditary component of this common diseaseand would also be valuable in the subsequent development of treatmentand management strategies for DD.

It will be appreciated from the above that there is a need to be able toassess individuals for a susceptibility for developing conditions atleast partially characterised by inappropriate scarring or fibrosis andalso to assess the prognosis for an individual suffering from such acondition. One way in which this may be approached is the identificationof polymorphisms or mutations in a gene that may be associated with aparticular medical condition. Once identified, such polymorphisms canprovide useful information to physicians who need to manage, treat orestablish a susceptibility to developing a condition. One way in whichthe information may be used is in the development of a genetic test.

Genetic testing may be defined as the analytical testing of a patient'snucleic acid to determine if the DNA of a patient contains mutations (orpolymorphisms) that either cause or increase susceptibility to acondition or are in association with the gene causing the condition andare thus potentially indicative of a predisposition to that condition.

The early detection of a predisposition to a condition presents the bestopportunity for medical intervention. Early genetic identification ofrisk may improve the prognosis for a patient through early interventionbefore clinical symptoms manifest.

In cases where patients with similar symptoms are treated with variablesuccess with the same therapeutics, genetic testing may differentiatepatients with a genetic rather than developmental basis for theirsymptoms, thus leading to the potential need for different approaches totherapy.

It is an aim of the present invention to provide methods for geneticscreening to indicate a risk or predisposition to conditions at leastpartially characterised by inappropriate fibrosis or scarring.

According to a first aspect of the present invention there is providedan in vitro method for diagnosing or detecting a predisposition to acondition at least partially characterised by inappropriate fibrosis orscarring, the method comprising examining the ZF9 gene, and regulatoryelements thereof, derived from a subject of interest to detect thepresence of a genetic polymorphism or mutation.

By “polymorphism” we mean a region of a gene, or regulating elementsthereof, where the nucleotide base sequence may vary betweenindividuals. There is often a predominant genotype which represents theusual form, or wild type, of a gene with subsets of a population havinga polymorphism which confers a different genotype. Certain polymorphismscan be prevalent in individuals of particular ethnic backgrounds, orfrom specific geographical areas. A polymorphism may not affect functionof the gene; may lead to differences in the function of the gene; mayproduce an inactive gene product; or may modulate the production of thegene product.

By “mutation” we mean a region of a gene, or regulating elementsthereof, where the nucleotide base sequence may vary from the wild type.The mutation may for example comprise a base substitution, deletion oraddition. Certain mutant forms of the gene can be prevalent inindividuals of particular ethnic backgrounds, or from specificgeographical areas. A mutation may not affect function of the gene; maylead to differences in the function of the gene; may produce an inactivegene product; or may modulate the production of the gene product.

By “gene” we mean all coding sequences between the start and stop codonof the Zf9 gene (including introns and exons).

By “regulatory elements” we mean the DNA that is 5′ and 3′ of the geneand which is involved in regulating gene transcription. For instance,transcription factor binding sequences, the TATA box, the 5′ promoterand 5′ and 3′ untranslated regions (UTRs).

The method according to the first aspect of the invention allows aninvestigator to identify a subject with a genetic polymorphism ormutation in the ZF9 gene or its regulatory elements to determine thosesubjects who have, or are more at risk of developing, a condition atleast partially characterised by inappropriate fibrosis or scarring.This allows for appropriate action to be taken to prevent or lessen thelikelihood of onset of the condition or to allow appropriate treatmentthereof. The method is also useful for establishing a prognosis for asubject that has already been diagnosed as suffering form a particularcondition.

The transcription factor Zf9 is one of a multitude of biologicalmolecules that is implicated in the pathophysiological processes thatlead to the development of a scar or fibrotic tissue. The gene for Zf9has been mapped to a locus on human chromosome 10p15. The sequence offull-length human (and rat) cDNAs has been identified by Ratziu et al.(Proc. Nat. Acad. Sci 95: 9500-9505, 1998) and the gene sequence of Zf9is publicly available as Gene Bank Accession No. AB017493.

Zf9 is also known as Core Promoter Element-Binding Protein (COPEB orCPBP); B Cell-derived Protooncogene 1 (BCD1); GC-Rich Sites BindingFactor (GBF); and Kmeppel-like Factor 6 (KLF6).

The inventors performed experiments to screen the Zf9 gene and itsregulatory elements for polymorphisms and mutations which may beassociated with a condition at least partially characterised byinappropriate fibrosis or scarring. Having established such anassociation the inventors have established that DNA taken from a subjectmay be analysed to help establish a diagnosis of the condition or toestablish whether or not a subject is predisposed to develop such acondition.

The condition may be a form of pathological scarring of the skin (e.g.hypertrophic scarring or keloids) or an internal scar or fibrosis asmentioned above. Alternatively the condition may be a fibrotic diseaseor disorder also as mentioned above. Other conditions that may bediagnosed or detected according to the method of the invention includefibrotic disorders of the skin such as:

Sclerodemia

Systemic sclerosis

Crest Syndrome

Tuberous sclerosis with skin patches

Familial cutaneous collagenoma

Metabolic and immunologic disorders of the skin (porphyria cutaneatarda, chronic graft versus host disease)

Eosinophilic facsitis

Discoid lupus erythematosus, Dermatomyositis

Mixed connective tissue disease

Drug-induced skin fibrosis—bleomysin, PVC, silicates

Peyronies

Oral submucous fibrosis

Fibrosis induced following dietary and environmental exposures.

Fibrotic disorders of other organs may also be detected or diagnosed.These include:

Pulmonary/cardiac fibrosis

Liver fibrosis/cirrhosis

Renal fibrosis

GI tract fibrosis

Drug induced fibrosis (e.g. post organ transplantation)

Central and peripheral nervous system fibrosis

Vascular system (veins and arteries) fibrosis

Male & female genitourinary tract fibrosis

Gynaecological (fallopian tube fibrosis, uterine fibromas)

The method of the first aspect of the invention is particularly suitedfor diagnosing or detecting a predisposition to Dupuytren's Disease.

The Zf9 gene to be examined is preferably derived from a human subject.However it will be appreciated that DNA derived from animal subjects ofvetinary interest may also be tested according to the method of theinvention.

Preferred polymorphisms and mutations which may be detected according tothe first aspect of the invention are located in the untranslated DNA 3′of the Zf9 coding sequence. A most preferred polymorphism is located atposition 1140 of Gene Bank Accession No. AB017493 (NCBI Assay Id (ss#)20354; and Reference SNP Id (rs#) 17731) and is referred to herein as“the 1140 polymorphism”.

This 1140 polymorphism represents a point mutation of a Guanosinenucleotide (G) to an Adenosine nucleotide (A). Genotypes AA, AG and GGwere identified for this polymorphism.

The point mutation is located 1022 bases from the start of the codingsequence of the Zf9 gene and is found in the 3′ untranslated region. Thetwo allelic forms have the following base sequence:

The “G” form: gtccagggtcacccacataccatgcaccacgggtgctatgcc [G] cttcttacagg(Seq ID No. 1) acctttttagccctcaaaagaccttccaaggagaggccctggaggcaactgggtagggtgcagaaac The “A” form: gtccagggtcacccacataccatgcaccacgggtgctatgcc [A]cttcttacagga (Seq ID No. 2)cctttttagccctcaaaagaccttccaaggagaggccctggaggcaactgggtaggg tgcagaaac

Initial experiments established that the GG and AG genotype issignificantly over-represented in subjects with Dupuytren's Disease (DD)whereas a higher proportion of control subjects were of an AA genotype.

Further work has established that subjects with other fibrotic disorders(e.g. scleroderma or renal fibrosis) and subjects liable to developsevere/pathological scarring (e.g. keloids) also have a high frequencyof the G allelle. Therefore the polymorphism may be examined accordingto the method of the invention for diagnosing or detecting apredisposition to a variety of conditions at least partiallycharacterised by inappropriate fibrosis or scarring.

Various techniques may be used to detect polymorphisms or mutationsaccording to the method of the invention.

A preferred technique involves Restriction Enzyme Digestion (RED) and isbased upon the fact that polymorphisms can lead to the production ofdifferent sized DNA fragments following treatment with a restrictionenzyme (because of the introduction or deletion of a restriction site bythe mutation causing the polymorphism). These fragments may bevisualised on gels and the polymorphism or mutant identified based uponthe number and size (i.e. distance moved on the gel) of the fragmentsfrom a DNA sample derived from a subject.

Preferably the DNA comprising the Zf9 gene and/or regulatory elementsthereof is amplified prior to detection of the polymorphism Thisamplification is preferably by means of the polymerase chain reaction(PCR). For instance a preferred method according to the invention knownas the PCR-restriction fragment length polymorphism method (PCR-RFLP) isdescribed in more detail in Example 1 and involves PCR amplification ofDNA containing the polymorphism prior to RED and subsequent analysis.

PCR primers need to be designed such that they are suitable foramplifying a region around the relevant polymorphism. Suitable primersfor amplifying the 1140 polymorphism are listed below as SEQ ID No. 3and SEQ ID No. 4.

Forward primer: 5′ GTCCAGGGTC ACCCACATAC 3′ (SEQ ID No. 3) Reverseprimer: 5′ GTTCTGCACC CTACCCAGTT 3′ (SEQ ID No. 4)

For some polymorphisms or mutations neither the wild type nor the mutantallele abolishes or introduces a restriction enzyme site. When this isthe case, a restriction enzyme site may be introduced by specificallydesigning PCR primers that introduce restriction sites into theamplified product. The introduced enzyme site allows differentiationbetween polymorphic alleles and wild type by size analysis. For exampleif the restriction products of the amplified product are analysed by gelelectrophoresis (agarose or polyacrylamide gel, for example) the alleleswith the introduced restriction enzyme site will produce an extra bandon the gel.

Other techniques that may be used to detect polymorphisms or mutationsaccording to the present invention include:

-   -   (1) Direct sequencing of the polymorphic region of interest        (e.g. using commercially available kits such as the Cy5™ Thermo        Sequenase™ dye terminator kit—Amersham Pharmacia Biotech);    -   (2) Sequence Specific Oligonucleotide Hybridization (SSO)        (involving dot or slot blotting of amplified DNA molecules        comprising the polymorphic region; hybridisation with labelled        probes which are designed to be specific for each polymorphic        variant; and detection of said labels);    -   (3) Heteroduplex and single-stranded conformation polymorphism        (SSCP) Analysis (involving analysis of electrophoresis band        patterns of denatured amplified DNA molecules comprising the        polymorphic region);    -   (4) Sequence Specific Priming (SSP) [also described as        Amplification Refractory Mutation System (ARMS)];

(5) Mutation Scanning [e.g. using the PASSPORTS™ Mutation Scanning Kit(Amersham Pharmacia Biotech)];

-   -   (6) Chemical Cleavage of Mismatch Analysis;    -   (7) Non-isotopic RNase Cleavage Assay (Ambion Ltd.);    -   (8) Enzyme Mismatch Cleavage Assay; and    -   (9) Single Nucleotide Extension Assay.

The method according to the first aspect of the invention isparticularly suitable for being carried out on genomic DNA, particularlyon isolated genomic DNA. Such genornic DNA may be isolated from blood ortissue samples (e.g. hair, oral buccal swabs, nail or skin) or fromother suitable sources using conventional methods. Preferably the DNA isisolated from whole blood or granulocytes.

A prediction or diagnosis based upon the method according to the presentinvention depends upon an association being made between a particularcondition and the specific polymorphism or mutant in question. Suchassociations were established by the inventors by performing furtherexperiments and making statistical analyses (e.g. see the Example).Association between the 1140 polymorphism are discussed above and in theExamples. Provision of data based upon association analyis enables aclinician to interpret the significance of genotypes identified bysequencing DNA according to the method of the invention. The clinicianmay then make a judgment regarding the likelihood of a patientdeveloping, or having, a particular disease or disorder. Such knowledgeis important in the clinical management of specific conditionsassociated with inappropriate scarring or fibrosis. It will beappreciated that data relating to the association of a particulargenotype with a condition may be provided to a user of the methodaccording to the invention (e.g. a technician or clinician) byincorporating a data sheet as part of a kit (see below).

Genetic testing may be carried out either pre-natally, peri-natally orpost-natally when it is desired to test whether or not a neonate orchild is likely to have inherited a predisposition to develop acondition at least partially characterised by inappropriate fibrosis orscarring. This is particularly useful when there is believed to be afamily history of developing the condition.

The test is particularly useful for testing subjects pre-operatively.The results of such a test are useful for establishing whether or notthere could be healing complications for the subject undergoing surgery(e.g. hypertrophic scarring, keloids or internal fibrosis/scarring).

The test is also useful before a therapeutic regimen is established fortreating a condition characterised by inappropriate scarring orfibrosis. The results of the test according to the invention may be usedby a clinician to help in the selection of medicaments used and thedosage thereof.

It is most preferred that the method of the invention is used to testsubjects with a family history of developing a condition at leastpartially characterised by inappropriate scarring or fibrosis.

The various elements required for a technician to perform the method ofthe first aspect of the invention may be incorporated in to a kit. Thusaccording to a second aspect of the present invention there is provideda kit comprising:

-   -   A) PCR primers for amplifying genetic polymorphisms in the ZF9        gene and regulatory elements thereof that are linked to a        condition characterised by inappropriate scaring or fibrosis;        and    -   B) Control DNA samples of known genotype for each polymorphism.

It is preferred that the kit comprises primers specific for a targetsequence of sample DNA known to contain a polymorphism of interest Forinstance, suitable PCR primers for a kit for genotyping the 1140polymorphism are the primers of SEQ ID No. 3 and SEQ ID No. 4.

The kit may farther comprise:

-   -   C) a suitable restriction enzyme for generating fragments of the        DNA sample;    -   D) a data card outlining linkage between a particular        polymorphism and a disease;    -   E) protocols for PCR amplification, restriction enzyme digestion        of PCR products and agarose gel electrophoresis of DNA        fragments;    -   F) relevant buffers.

Buffers provided with the Kit may be in liquid form and preferablyprovided as pre-measured aliquots. Alternatively the buffers may be inconcentrated (or even powder form) for diluting.

The Kit may further comprise suitable reaction vessels, centrifuge tubesetc.

A multitude of biological molecules are involved in the fibrotic orscarring process and it would be expected that many different factorslead to the development of a scar or fibrosis. The growth factor TGF-β1is an example of a biological molecule which has been implicated as amajor factor in the development of scars and fibrotic tissues. TheTGF-β1 gene is known to be polyniorphic and several polymorphisms of theTGF-β1 gene have been reported in the literature. The inventors exploredthe hypothesis that there is an association between four known TGF-β1polymorphisms and the fibrotic condition Dupuytren's disease. TGF-β1genotyping was performed in Caucasian individuals with DD and comparedwith a control Caucasian population These studies established that therewas a negative association between the Caucasian controls andDupuytren's disease cases for the TGF-β1 polymorphisms. The fact thatpolymorphisms in a fibrotic growth factor such as TGF-β1 did not exhibitany association with a condition characterised by inappropriate scarringor fibrosis, illustrates how surprising the inventors' discovery wasthat the polymorphisms in the gene for Zf9 showed such significantassociation.

The invention will be further described, by way of example only, withreference to the accompanying drawing, in which:

FIG. 1 is an electropherogram illustrating the polymorphism at position1140 Gene Accession No. AB017493; and

FIG. 2 is a photograph of a gel illustrating the difference in size ofamplified DNA fragments corresponding to the genotypes GG, AG and AA inExample 1

EXAMPLE 1 Linkage Analysis of a ZF9 Polymorphism

Samples were taken from a group of patients with Dupuytren's Disease(DD) and a control group to investigate the correlation between a Zf9polymorphism and the condition.

1.1. Patients & Methods

1.1.1 Patients

Dupuytren's patients (n=138) were entered into the study (119 men and 19women). The age range was between 37 to 90 years with a mean age of 55.8years. Cases were all unrelated Caucasians from the Northwest region ofEngland, U.K. Dupuytren's cases were identified through operative recordclinical codes from the South Manchester University Hospital Trust andWrightington Hospital in the North West region. All patients were seenby a medically qualified person who took a full medical history using aproforma and examined both hands of each case. All cases had confirmeddiagnosis of Dupuytren's disease pre-operatively with the presence ofcharacteristic dupuytren's nodules in the palm of the hand and/or digitswith or without contracture of either the MCPJ or the PIPJ.

A control group (n=161) comprised ethnically matched healthy Caucasianmen and women and was selected from general practice registers.

The local and hospital ethical committees had given approval for thestudy protocol and profomias. Written consent was obtained from allindividuals.

1.1.2 DNA Extraction Method

Blood samples were collected from subjects using standard techniques (15ml of venous blood were collected).

DNA was extracted from peripheral blood cells using a commerciallyavailable DNA extraction kit (Qiagen,UK). DNA concentrations weremeasured and diluted in buffer to 100 ng/μl using sterile, Qiagenbuffer.

1.1.3 Genotyping Method

Zf9 genotyping was carried out using the polymerase chainreaction-restriction fragment length polymorphism (PCR-RFLP) method.

The polymorphism chosen for the study was found to be in the 3′untranslated region of the ZF9 gene and corresponded to the 1140polymorphism described above (NCBI Assay Id(ss#): 20354 Reference SNPId(rs#): 17731).

PCRs were carried out in 96 well microlitre plates. Each PCR consistedof 1 μl of DNA (100 ng/μl ), 2.5 μl of x1 NH₄ buffer (Bioline), 2.5 μlof each 2 mM d'NTP (Behring), 0.1 μl of 0.1 unit Taq polyrnerase(Bioline), 6 μl of Betaine, 0.75 μl MgCl₂, 0.1 μl each of 2.5 pmolforward and reverse primer of Sequence I.D. No 2 and 3 respectively andmade up to 25 μl reaction mix with autoclaved, distilled water. Thesequences of the primers used are given below:

Forward GTCCAGGGTC ACCCACATAC; (Seq I.D. No. 3) Primer: and BackwardGTTCTGCACC CTACCCAGTT (Seq I.D. No. 4) Primer:

PCR was carried out under the following conditions: 2 minutes ofdenaturation at 95° C.; followed by 35 cycles of further denaturation of45 seconds at 95° C.; then 1 minute at either annealing temperature (aslisted in table 1), and 45 seconds of extension at 72° C.; a finalelongation step of 5 minutes at 72° C. was included.

The PCR-RFLP conditions was as follows: Amplified DNA (5 μl) wasdigested with the appropriate enzyme (0.8 μl) including buffer (1 μl)and made up to a 10 μl reaction mixture using distilled water. Digestionwas carried out overnight in a Hybaid Omnigene thermal cycler. Theenzyme used was Aci I (recognition sequence ccgc). Enzyme was purchasedfrom the New England Biolabs. The digested products were fractionated in4% polyacrylamide gels and visualised by ethidium bromide andultraviolet light using standard procedures.

1.1.4 Statistical Analysis

Association with DD was investigated by comparing the distribution ofits allele frequencies between DD patients and controls using a singleglobal Pearson's chi-squared test. STATA 6 statistical data analysisprogramme was used to calculate p values and odds ratios.

1.2 Results

The results of an electropherogram of the region surrounding the 1140region of Genbank Accession No. AB017493 are presented in FIG. 1 andconfirm the existence of the G/A polymorphism.

Having confirmed the existence of the polymorphism, DNA Fragmentsgenerated according to method 1.1.3 were run on polyacrylarnide gels asspecified. FIG. 2 illustrates that different genotypes generated DNAfragments of a unique size which could easily be differentiated byreading the gel. Accordingly gels were generated for each individual inboth the DD group and control group and statistical analysis made of anylinkage between genotype and health status.

Statistical analysis of the genotyped samples are provided in Table 1.These data illustrate that the GG and AG genotype is significantlyover-represented in subjects with Dupuytren's Disease (DD) whereas ahigher proportion of control subjects were of an AA genotype.

Accordingly a linkage between the GG and AG genotypes of the 1140polymorphism in the 3′ untranslated region of the Zf9 gene and DD wasestablished. It will be appreciated that such a linkage will also existwith other conditions characterised by inappropriate scarring orfibrosis.

Table 1: Zf9 genotype & allele frequencies

DD Cases CONTROL (n = 138) (n = 161) Allele frequency 1 (A) 84 (30%) 128(40%) 2 (G) 192 (70%) 194 (60%) Genotype frequency 1 (A/A) 13 (9%) 23(14%) 2 (A/G) 58 (42%) 82 (51%) 3 (G/G) 67 (49%) 56 (35%) p-value =0.046

EXAMPLE 2

Having established a correlation between a condition characterised byinappropriate scarring or fibrosis and a genotype conferred by the 1140polymorphism for ZF9, the methodology of Example 1 was repeated toscreen subjects according to the method of the first aspect of theinvention to establish the genotype of the subject.

The results of the genetic test could then be used by a clinician toprovide medical advice to the individuals. For instance, asymptomaticindividuals found to have the G allelle may be advised that they were atincreased risk of developing a condition characterised by inappropriatescarring or fibrosis. If appropriate, such individuals could be advisedto make life style changes and/or receive prophylactic treatment.Individuals having the G allelle, who are already suffering from acondition characterised by inappropriate scarring or fibrosis, may beadvised to adjust their medication or habits as they are potentially atrisk of developing a more severe form of the condition.

1. An in vitro method for detecting a predisposition to a condition atleast partially characterised by pathological or excessive formation offibrotic tissue, the method comprising i) obtaining DNA comprising theZf9 gene from a subject of interest, wherein said Zf9 gene comprises thesequence of SEQ ID NO:1 or the sequence of SEQ ID NO:2 and whereinnucleotide 43 of SEQ ID NO:1 and SEQ ID NO:2 is a polymorphic site, ii)amplifying a region of said Zf9 gene in said DNA comprising saidpolymorphic site, and iii) analyzing the amplification product resultingfrom step (ii) for the presence of guanosine at said polymorphic site,wherein the presence of guanosine at said polymorphic site indicatessaid subject is predisposed to said condition.
 2. The method accordingto claim 1 wherein, in step (ii), a region comprising SEQ ID NO:1 isamplified.
 3. The method according to claim 1 wherein the condition isDupuytren's Disease.
 4. A method according to claim 1 wherein the ZF9gene, and regulatory elements thereof, is derived from a sample ofgenomic DNA.
 5. A method according to claim 4 wherein the genomic DNA isisolated from blood or tissue samples.
 6. A method according to claim 1wherein, in step (ii), said region is amplified using polymerase chainreaction (PCR) primers.
 7. A method according to claim 6 wherein saidPCR primers are: Forward primer: 5′ GTCCAGGGTC ACCCACATAC 3′ (SEQ ID No.3); and Reverse primer: 5′ GTTCTGCACC CTACCCAGTT 3′ (SEQ ID No. 4).


8. A method according to claim 1 wherein said analyzing step (iii) iseffected by restriction digestion and size analysis.